Targeted Therapy
Targeted drug delivery is a powerful technology which holds numerous potential advantages over conventional drug formulations. Methods for controlling the action of a drug in a specific temporal and spatial distribution, so as to direct its action to the desired target organ or cell, are currently being investigated to achieve increased safety and decreased side effects, increased bioavailability of the drug, improved dosage forms and administration schedules, and overall increased efficiency of the treatment. Since the development of methods of producing monoclonal antibodies (mAbs) by
Kδhler and Milstein, and the initial clinical trials of antibody therapy in cancer patients, there has been progress in antibody-based therapeutics, particularly in oncology. Early clinical trials with murine achieved biological effects principally by inducing immune effector functions (complement-dependent cytotoxicity, and antibody-dependent cellular cytotoxicity), which had the potential for therapeutic effect with minimal toxicity, as compared with standard chemotherapy. The range of possible therapeutic approaches with mAb targeted therapy was similarly impressive, with the possible selective delivery of isotopes, drugs, toxins and other cytotoxic materials to tumors by linking these agents to tumor targeting mAbs. Subsequent trials in a wide range of tumor systems with numerous constructs, however, demonstrated the limitations of murine antibodies regarding immunogenicity and the development of human anti-mouse antibody responses, (White et al., 2001) and demonstrated that the effective targeting and treatment of most solid tumors may not be achieved successfully with murine constructs.
Recent developments in the fields of protein engineering and particularly in antibody engineering techniques have led to the production of improved targeting moieties, such as humanized mAb constructs, recombinant antibody fragments such as single-chain antibodies (scFvs), short peptides, non-antibody ligand-binding proteins and even non proteinaceous molecules such as carbohydrates. Still, targeted therapy is mostly focused on cancer, with limited attention to immune system and autoimmune disorders.
Immunoconjugates are bifunctional molecules that consist of a “targeting” domain that localizes in tumors coupled to a therapeutic moiety (Benhar and Pastan, 1997). Immunoconjugates, in the broadest definition, may utilize mAb, mAb fragments, hormones, peptides or growth factors to selectively localize cytotoxic drugs, plant and bacterial toxins, enzymes, radionuclide, photosensitizers, or cytokines to antigens expressed on tumor cells or on cells of the tumor neovasculature (White et al., 2001).
Another approach for controlling drug delivery includes targeted drug activation, i.e. the use of prodrugs, wherein the drug composition is inactive when administered, but is converted to active form in vivo through exposure to a particular physiologic environment or metabolic process. In some cases, the drug is linked to a masking moiety rendering it inactive, by means of a linker that can be, for example, acid-labile or enzyme-cleavable (Ulbrich and Subr, 2004). Examples for the use of this approach are WO 00/33888 and US 2004/014652.
Other approaches for controlling drug delivery include the use of carriers, such as liposomes, microspheres and dendrimers, to alter the physico-chemical qualities of a drug, thus controlling its distribution and half-life.
These and other studies possess several advantages over the use of traditional drug formulations, but each methodology is limited by its own shortcomings, aside from technical challenges related to tissue penetration, stability and immunogenicity. For example, the extent to which drug disposition can be controlled through nonselective mechanisms, such as carriers and prodrugs, is limited. In addition, the drug carrying capacity of existing targeted therapies is a key issue in their potency, as some of the putative targeted molecules are only expressed in a limited copy number on the target cell.
Thus, attempts at providing targeted drug delivery methods combining the benefits of the different approaches are being made. For example, conjugation schemes, such as the use of dendrimers and branched linkers were devised to maximize the drug payload per targeting molecule that binds a target site. Other approaches include directed micelles, liposomes or polymers (see, for example, WO 03/035611, WO 00/53236, US 2004/11648 and U.S. Pat. No. 6,387,397), some of them utilizing viral components for target recognition and/or structural purposes (Felnerova et al., 2004, Garcea et al., 2004). However, despite the recent advances made in the field of targeted drug delivery, the need remains for developing new effective target-specific drug delivery methods.
Drug Resistant Bacteria
The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels, thus necessitating the strong need to develop new antimicrobial agents. The use of antimicrobial drugs for prophylactic or therapeutic purposes in humans, or for veterinary or agricultural purposes, has provided the selective pressure favoring the survival and spread of resistant organisms. Because of more intensive antibiotic use in hospitals as compared with the community, higher rates of resistance are noted in hospital pathogens, especially in the intensive care unit where infections caused by Gram-positive bacteria are increasing.
Attempts at discovering totally new agents effective in the treatment of bacterial disease caused by resistant organisms are currently being made. The large majority of marketed antibacterials have been targeted against the bacterial cell wall or macromolecular biosynthesis (DNA, RNA or protein). Bacteria have, however, developed defenses, either by mutation or by acquiring new genes from other bacteria.
These defenses have included acquiring enzymes to deactivate the drugs, alteration in permeability of cell walls, efflux proteins to pump antibacterial rapidly out of cells and alteration of target molecules in order to protect them against attack by antibacterials. However, for the first time in several years, new classes of compounds designed to avoid defined resistance mechanisms are undergoing clinical evaluation. Classical short-term approaches include chemical modification of existing agents to improve potency or spectrum. Long-term approaches are relying on bacterial and phage genomics to discover new antibiotics that attack new protein targets which are essential to bacterial survival and therefore with no known resistance. The rationale of such approaches is that the only certain way to avoid encountering previously generated resistance is to seek new antibiotic targets.
In both traditional and newly developed antibiotics, the target selectivity lies in the drug itself, in its ability to affect a mechanism that is unique to the target microorganism and absent in its host. As a result, a vast number of potent drugs have been excluded from use as therapeutics due to low selectivity, i.e. toxicity to the host as well as to the pathogen.
Therapeutic Use of Bacteriophages
Bacteriophages (phages) are a phylum of viruses that infect bacteria, and are distinct from plant or animal viruses. Upon infection of a corresponding host bacterium, the phages may undergo a “lytic” life cycle, a “lysogenic” life cycle that can potentially become lytic, or a “non-lytic” life cycle. Virulent Phages replicate through the lytic cycle, causing lysis of the host bacterial cell as a normal part of their life cycles. Temperate phages can undergo either a “lysogenic” life cycle, in which the phage may be integrated into the bacterial host DNA to persist as a prophage, or replicate by means of the lytic life cycle and cause lysis of the host bacterium. The natural capability of phages to selectively infect a host bacterium and to kill it is the basic concept upon which “phage therapy” was built. Phage therapy was pioneered by D'herelle, who recognized bacteriophages as epizootic infections of bacteria Phages were almost immediately deployed for antibacterial therapy and prophylaxis by D'herelle, and later on by others. Although many early phage therapy trials were reported successful, and many of the major pharmaceutical firms sold phage preparations (e.g., Parke-Davis and Lilly in the United States), there were also failures. The early trials of bacteriophage therapy for infectious diseases were thus confounded, because the biological nature of bacteriophage was poorly understood. Therefore, the advent of antibiotics resulted in the absence of rigorous evaluations of phage therapy until very recently. However, recent laboratory and animal studies, exploiting current understandings of phage biology, suggest that phages may be useful as antibacterial agents in certain conditions (reviewed in Summers, 2001).
The therapeutic application of phages as antibacterial agents is still impeded by several factors: (i) the failure to recognize the relatively narrow host range of phages; (ii) the presence of toxins in crude phage lysates; and (iii) a lack of appreciation for the capacity of mammalian host defense systems, particularly the organs of the reticuloendothelial system, to remove phage particles from the circulatory system (Merril et al., 1996). Solutions to the latter problem were proposed in the form of selecting long-circulating phage mutants that escape or even repel the host defense system (Merril et al., 1996) or modifying the phage coat with i.e. polyethylene glycol to “shield it” from host defense surveillance mechanisms (US patent application 2004/0161431).
To date, most attempts at anti bacterial phage therapy still rely on the exquisite specificity of phages to infect (and kill) a unique host bacterium (for example U.S. Pat. No. 6,485,902, PCT application WO 2004/052274 and references therein), although a few attempts at creating bacteriophages with a broader host spectrum as an anti-bacterial therapy have been reported (US 2002/0044922 and US 2003/216338).
WO 2004/062677 discloses compositions for treating a bacterial biofilm, comprising a first bacteriophage that is capable of infecting a bacterium within said biofilm, and a first polysaccharide lyase enzyme that is capable of degrading a polysaccharide within said biofilm. The composition preferably further comprises a pharmaceutically-acceptable antimicrobial agent, and may also include a DNase.
The growing use of bacteriophages as molecular biology tools has led to the development of another therapeutic use for phages, namely phage-mediated gene delivery to mammalian cells. This technology was developed following discoveries by several groups that identified “internalizing phages” while searching for cell-surface molecules that could be used as targets in targeted immunotherapy. Antibodies that bind cell surface receptors in a manner whereby they are endocytosed are useful molecules for the delivery of drugs, toxins, or DNA into the cytosol of mammalian cells for therapeutic applications. Traditionally, internalizing antibodies have been identified by screening hybridomas. Several groups turned to phage display as an alternative approach. In a pioneering work from the Marks group, an anti ErbB2 antibody was used to determine the feasibility of directly selecting internalizing antibodies from phage libraries and to identify the most efficient display format. Using a known antibody in a scFv format, displayed monovalently on a phagemid, they demonstrated that anti-ErbB2 phage antibodies can undergo receptor-mediated endocytosis. This study also defined the role of affinity, valency, and display format on the efficiency of phage endocytosis and identified the factors that led to the greatest enrichment for internalization. This work demonstrated that phages displaying bivalent scFvs (diabodies) or multiple copies of the scFv were more efficiently endocytosed than phage displaying monomeric scFv (Becerril et al., 1999).
Subsequent studies have further demonstrated the use of internalizing phages as gene delivery vessels. Vectors based on filamentous phages, lambdoid phages or phagemids, carrying antibodies or ligands of mammalian internalizing cell surface receptors, have been reported (see, for example, Kassner et al., 1999, Poul and Marks, 1999, Larocca and Baird, 2001, Larocca et al., 2001, Urbanelli et al., 2001, U.S. Pat. Nos. 6,451,527, 6,448,083, and International Application WO 98/05344).
Additional phage-based therapeutic approaches use whole phage particles for vaccination. Initially, phage lysates were used as immunogens with the aim of eliciting immune response against components of the phage bacterial host that are present in the lysate. Following the utilization of phage display for the identification of antibody epitopes and mimotopes, several groups used the peptide-displaying phages as immunogens to elicit an immune response against the original pathogen or diseased tissue from which the epitope originated, which met with limited success (Meola et al., 1995; Bastien et al., 1997; Delmastro et al., 1997; Phalipon et al., 1997; Menendez et al., 2001). With the advent of DNA vaccination, phages were adapted as carriers of DNA for similar purposes. This fledgling field was developed as an outgrowth of phage-mediated gene delivery approaches following the identification of cell-internalizing phages (Becerril et al., 1999; Poul and Marks, 1999; Poul et al., 2000). Recent studies have shown that bacteriophage-mediated DNA vaccination consistently gave better antibody responses when compared to naked DNA. Although a strong antibody response was also seen against the carrier phage coat proteins, this may actually enhance the efficiency of the delivery system, since the formation of immune complexes should more effectively target APCs following boosting (Clark and March, 2004, WO 02/076498, US 2004/0121974).
Several reports disclose bacteriophages linked to haptens for experimental purposes. For example, Haimovich and colleagues (Haimovich and Sela, 1969; Sulica et al., 1971) report the generation of protein-bacteriophage conjugates useful in detection of antibodies and antigens. These studies did not teach or disclose pharmaceutical compositions comprising protein-bacteriophage conjugates.
Other studies disclose particles of bacteriophage components that contain or enclose drugs for therapeutic uses. Brown et al. (2002) discloses a virus-like particle comprising recombinant MS2 coat proteins and a toxin molecule conjugated to an RNA molecule. U.S. Pat. No. 6,159,728 discloses a delivery system comprising a capsid formed from a coat protein of a bacteriophage selected from the group consisting of MS-2, R17, fr, GA, Qβ, and SP and a foreign moiety enclosed in the capsid, wherein the foreign moiety is of a size sufficiently small to be enclosed in the capsid and wherein the foreign moiety is linked to a RNA sequence comprising a translational operator of the bacteriophage, which translational operator binds to the coat protein during formation of the capsid.
However, none of the background art discloses pharmaceutical compositions having high drug-binding ratios on bacteriophage-based drug carriers suitable for targeted drug delivery. The development of an efficient, high-capacity system for drug delivery would be highly advantageous in the treatment of various diseases, such as cancer, bacterial and viral infections and any disease expressing specific and targetable markers.